Polony Sequencing Protocols

Paired-End-Tag (PET) Genomic Shotgun Library

Polony genomic resequencing is generally performed on a shotgun paired-tag library
(Shendure et al., 2005). Each library molecule is 135 bp in length, and has two 17- to
18-bp paired genomic tags separated and flanked by common sequences.
The key step in this protocol is the circularization of randomly sheared and size-selected
genomic DNA around a synthetic insert-linker. This links sequences that are separated
on the genome by a defined distance distribution.

1. Shear 15 μg genomic DNA to desired size distribution.
The Hydroshear (http://www.genomicsolutions.com) is the recommended instrument for
this purpose since, when the manufacturer’s instructions are followed, it produces a
population of fragments having a relatively tight size distribution and ends that can be
efficiently blunted. The authors typically shear DNA at a concentration of ∼60 ng/μl.

2. Purify DNA on Qiaquick columns (≤10 μg per column) as per manufacturer’s
instructions.
Use as few columns as possible.
3. To repair the DNA ends (i.e., blunt them following shearing), combine the DNA with
the components of the End-It DNA end repair kit as follows:

Incubate 30 min at 70◦C then cool to 4◦C. Transfer to ice.
In the A-tailing reaction, Taq DNA Polymerase adds a single A to the 3_ ends of the blunted
DNA in a template-independent fashion.

8. Extract DNA from the reaction mixture with phenol/chloroform and precipitate in
ethanol using 20 μg glycogen as carrier (UNIT 2.1A). Resuspend pellet in 40 μl buffer
EB.
Glycogen should be used as a carrier in all ethanol precipitation steps in this protocol.

10. Cut out the desired bands and extract DNA using the crush-and-soak method (UNIT 2.7).
Precipitate in ethanol using glycogen as a carrier, and resuspend in 10 μl buffer EB.
For a paired-tag library with an average intertag distance of 1 kb, excise and pool gel
bands centered at 1 kb with a total width of no more than 500 bp. Exposure of the DNA
to UV light during visualization should be minimized.

11. Quantitate the size-selected DNA by separating on an agarose or polyacrylamide gel
(link) and comparing the brightness of the smear to that of a known amount
of a molecular weight standard.
The concentration should be ∼20 to 40 ng/μl.

Circularize genomic material around synthetic oligonucleotides

12. Anneal oligonucleotides T30-T and T30-B in a thermal cycler by combining equal
amounts and heating to 95◦C for 10 min and then slowly cooling to room temperature
over the course of 1 hr.
The final concentration of the T30 insert-linker should be 100 μM. T30 has T 3_ overhangs
designed to be complementary to the A 3_ overhangs of the genomic DNA.

13. Ligate genomic DNA fragments in the presence of annealed T30 using the Quick
ligation kit as follows:

170 ng size-selected DNA (0.25 pmol at ∼1 kb)

0.8 μl 1 μM annealed T30 (0.8 pmol)

40 μl 2× Rapid Ligation Buffer

4.0 μl T4 DNA Ligase

H2O to a final volume of 80 μl.

Incubate reaction 10 min at room temperature then heat-inactivate 10 min at 65◦C.
Use the heat-inactivated reaction mixture directly in the next step without further
purification.

The ligation should be performed under conditions favoring formation of monomeric
recombinant circles (i.e., genomic DNA–T30). To this end, T30 should be present at a
three-fold molar excess to genomic fragments.

14. Eliminate all noncircularized material by exonucleolysis as follows:

1.0 μl 20 U/μl Exonuclease I

0.1 μl 100 U/μl Exonuclease III

80 μl heat-inactivated reaction mix

TE buffer, pH 8.0, to a final volume of 90 μl.
Incubate 45 min at 37◦C, and then heat-inactivate 20 min at 80◦C. Use this reaction
mixture directly in the next step without further purification.

Amplify circular DNA

15. Prepare the master mix for hyper-branched rolling-circle amplification of the circular
DNA:

12 μl 25 mM dNTP mix

30 μl 10× phi29 reaction buffer

15 μl 1 mM N6 oligonucleotides

213 μl H2O

30 μl circularized DNA.

Split into 6 tubes of 50 μl each.

16. Denature material by heating to 95◦C for 5 min, then anneal by rapidly cooling to
4◦C. Add 2.5 μl phi29 polymerase to each tube, keeping on ice. Incubate overnight
at 30◦C.

17. Pool amplified circular DNA into a single tube. Purify DNA using a Microcon-30
column according to the manufacturer’s instructions, washing with 1 ml TE buffer,
pH 8.0. Wash the membrane several times to maximize recovery.

18. Resuspend the DNA using 750 μl buffer EB, preheated to 50◦C. Quantitate the DNA
using a NanoDrop ND-1000 spectrophotometer.
Concentration should be ∼230 ng/μl.

Split into eight tubes of 125 μl each on ice, and then incubate at 37◦C for 30 min.
MmeI cuts 18 to 19 bp from its recognition site, which is 1 bp from the start of the genomic
fragment. The released fragments contain the common T30 sequence flanked by two 17-
to 18-bp tags of genomic DNA.

20. Immediately extract the DNA with phenol/chloroform and precipitate with ethanol
using glycogen as a carrier. Resuspend pellet in a total volume of 80 μl TE buffer,
pH 8.0.

21. Purify the 70-bp paired-insert library as in steps 9 and 10, resuspending precipitated
DNA in 20 μl TE buffer, pH 8.0.
The sample should be split across four 0.5-cm lanes of a precast 6% polyacrylamide gel
in TBE buffer.

25. Prepare emulsion PCR amplification primer sequences by annealing oligonucleotides
FDV-T and FDV-B and oligonucleotides RDV-T and RDV-B as in step 12.
The final concentration of each oligonucleotide should be 50 μM. FDV and RDV are not
5'-phosphorylated, so they will not ligate to one another.

26. Prepare the ligation reaction mixture by mixing the following components on ice:

8.0 μl blunt-ended library DNA (∼100 ng, 2 pmol)

1.0 μl 50 μM FDV (50 pmol)

1.0 μl 50 μM RDV (50 pmol)

2.5 μl 10× T4 DNA ligase buffer (∼0.5×)

21.1 μl 40% PEG

12.3 μl H2O.

27. Mix on ice, then add 2.0 μl of 2000 U/μl T4 DNA ligase and incubate overnight at
16◦C to ligate primer sequences to the library molecules.
After ligation, several species will be present: FDV-insert-FDV, FDV-insert-RDV, and
RDV-insert-RDV.

28. Increase volume to 100 μl with TE buffer, pH 8.0. Extract the DNA with phenol/
chloroform, precipitate in ethanol using glycogen as a carrier, and resuspend in
10 μl buffer EB.

Emulsion breaking protocol

This protocol is performed on a 96‐well microwell plate of emulsion following PCR, ~50ul of emulsion
per well.

1. Using a multichannel pipette add 100 uL of isopropanol to each well.

2. Pipette up and down at least 10 times

3. Using the multichannel pipette pool the isopropanol/PCR mixes into a reservoir

4. Transfer the pooled mixture into a 15 mL polypropylene falcon tube

5. Vortex for 1 min

6. Spin for 30 sec at 4000 rpm in table‐top centrifuge

7. Remove supernatant by decanting

8. Add 5 mL of isopropanol and resuspend the pellet via pipetting. Pipette for at least 1 min. Be
sure no clumps are visible.

9. Add 5 mL of isopropanol

10. Vortex for 30 sec

11. Spin for 30 sec at 4000 rpm in table‐top centrifuge

12. Remove supernatant making sure that you remove as much of the isopropanol as possible.

13. Resuspend the pellet in 6 mL of NXS. Pipette for ~ 1 min using pipetaid. The goal here is to make
sure that the pellet is VERY well resuspended. There cannot be any yellowish/orangeish clumps
(Even on the side of the tube). This is absolutely critical.

14. Vortex for 30 sec

15. Spin for 30 sec at 3000 rpm in table‐top centrifuge

16. Remove supernatant using magnetic particle separator

17. Resuspend in 1 mL of NXS and pipette for ~1 minute and then transfer to a 1.5 mL siliconized
eppendorf

Bead Capping Protocol

Church Lab 01‐09‐07

The purpose of this protocol is to attach a ‘capping’ oligonucleotide to every 3’ end on the bead –
unextended forward ePCR primers and 3’ template ends. The cap we use is an amino group, which
blocks any further ligation and also allows for coupling directly to the glass coverslip through a dual‐NHS
moiety (BS3).

11. Remove liquid from beads with MPC, quickly resuspend in 35 ul BS3 solution, and pipette into inlet
port of correct chamber on flowcell. It should only take several seconds to resuspend the beads and
transfer them to the flowcell chamber.

12. Leave flowcell upside-down on benchtop, un-disturbed, for 1 hour. During this time, the beads will
settle to the glass and be covalently coupled to it by the BS3.

Bead Enrichment

Church Lab 10‐Dec-2007

The purpose of this protocol is to enrich for amplified beads from a pool containing ~40% amplified
beads (the rest of which are unamplified). The protocol is similar to that in the Redbook except for the
volumes used and the ratio of capture beads to amplified ePCR beads. When performing this on a
sample to be arrayed in a gel‐less array, at least 2.5 plates of beads amplified to 40% should be used.

Add 40ul capture‐ePCR bead mix to top of glycerol. Immediately centrifuge at >= 16,000rcf for

1 min at RT

Pipette off all supernatant, being careful to not disturb the pellet. Transfer this supernatant

(containing ePCR‐capture bead complexes) to a new siliconized tube

Add 1ml dH2O and vortex. Centrifuge for 1 min at >= 16,000 rcf

Place on magnet, and remove most liquid (being careful to not aspirate any beads)

Add 200ul 0.1M NaOH, and vortex

Place on magnet and wait until solution turns pure white (all ePCR beads will now be against the

back of the tube facing the magnet)

Remove liquid and wash 2x w/ 200ul 0.1M NaOH, then transfer to a new tube and wash 2x w/

TE. Resuspend in 20ul TE. If enriching more than 1 plate, beads can be pooled into a single tube
when transferred after final wash with 0.1M NaOH.

Aminosilane treatment of coverslips

Church Lab 10‐Dec-2007

This protocol is used to wash and aminosilane‐treat the rectangular coverslips used in polony
sequencing. It is critical that the protocol be followed exactly as written to ensure 1) no fluorescent
contamination is present on the glass, and 2) coverslip is uniformly amino‐functionalized.

1. Load coverslips into rack (available from flowcell supplier)

2. Immerse coverslips in solution of 0.5% Triton X‐100 in dH2O. Wash by manually agitating the
rack for ~1 minute.

3. Pour all liquid off and wash thoroughly under running water to remove all detergent. Rinse with
dH2O.

4. Pour off all liquid and dry by immersion in 100% EtOH. Remove from EtOH and allow all ethanol
to evaporate (aided by use of a Dust‐off compressed air canister if possible).

6. Immerse rack of completely dry (and ethanol‐free) coverslips in silane solution for 30 seconds.
Use the handle to manually agitate the rack.

7. Remove and immediately immerse in fresh dry acetone rinse for 1 minute. Use the handle to
manually agitate the rack.

8. Remove rack and blow‐dry partially with compressed air (to remove the bulk of the acetone).

9. Completely air‐dry and store under vacuum at RT.

10. When cleaning beakers containing acetone and aminopropyltriethoxysilane, be sure to rinse
with fresh acetone to remove all traces of aminopropyltriethoxysilane. If present, it will form a
white precipitate upon evaporation of acetone.

Polony Sequencing Fluorescent Nonamer Sequences

Church Lab 10‐Dec-2007

All nonamers are synthesized by IDT on the 1 umol scale, with HPLC purification. Specify N:25:25:25:25
for all degenerate bases. Average yield is ~30 nmol for Cy3, Cy5, and TxRed, ~100 nmol for FAM.